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Activated leukocyte composition and uses for wound healing

a technology of activated leukocytes and compositions, which is applied in the direction of drug compositions, biocide, bandages, etc., can solve the problems of decreased quality of life, increased morbidity and mortality, etc., and achieves the effect of increasing the yield and viability of leukocytes (wbcs) and a higher percentage of activated granulocytes

Inactive Publication Date: 2013-03-21
MACROCURE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent invention has been found to have several benefits over existing wound healing products that contain white blood cells. It has a higher yield and viability of leukocytes, a higher percentage of activated granulocytes, and unexpectedly high concentrations of mesenchymal stem cells and endothelial progenitor cells. It also has a relatively high percentage of activated monocytes and a relatively higher percentage of CD8 T-cells compared to CD4 T-cells. Overall, this invention has been shown to have improved characteristics for wound healing compared to existing products.

Problems solved by technology

Complications from wounds may result in increased morbidity and mortality (Doshi, 2008).
These wounds lead to a decrease in quality of life and can result in increased morbidity and mortality.

Method used

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  • Activated leukocyte composition and uses for wound healing
  • Activated leukocyte composition and uses for wound healing
  • Activated leukocyte composition and uses for wound healing

Examples

Experimental program
Comparison scheme
Effect test

example 1

Analysis of Cellular Activation

[0074]An activated leukocyte composition made in accordance with the preferred embodiment of the present invention was quantified by the analysis of various cell surface markers. An increase in platelet interaction with either monocytes or granulocytes through the expression of P selectin is a sign of activation of the monocytes and granulocytes. Because residual platelets remaining in the buffy coat adhere to activated granulocytes and monocytes the latter exhibit platelet marker CD42b on their surface.

[0075]CD62L is an adhesion receptor from a selectin family. It is constitutively expressed on all classes of leukocytes including granulocytes, monocytes and lymphocytes. Upon activation, leukocytes rapidly shed off CD62L from their surface. CD62L is a plasma membrane protein which is shed during activation and thus decreases with cell activation. CD42b is a platelet activation marker involved in the process of coagulation as an aggregating factor. It i...

example 2

Analysis of an Activated Leukocyte Composition

[0081]Tables 3 and 4 depict the cellular compositions of the final ALC harvested at the end of production as determined by analysis with a Cell Dyn analyzer. Cells were analyzed (after activated leukocytes were re-suspending suspended the activated leukocytes in serum) was analyzed in triplicate with an automatic Cell Dyn analyzer. In addition cell viability was confirmed by Viable cells were stained using trypan blue exclusion and observed under a microscope. Tables 3 and 4 summarize the cellular composition of 8 batches of the final ALC.

TABLE 3Composition of Activated Leukocyte CompositionPlateletsErythrocytesLeukocytes(103 / μl)(106 / μl)(103 / μl)Concentration46.80.1 6.8in final ALCStandard39.20.063.8Deviation

TABLE 4Leukocyte Composition in ALCLeukocytesGranulocytes NeutrophilsBasophilsEosinophilsMonocytesLympho-%%%%cytes %% in final65.5 1.64.69.118.5 ALCStandard8.20.33  2.14.1DeviationRange52-781-21-96-1213-24

example 3

Analysis of an Activated Leukocyte Composition

[0082]Leukocytes were sampled at three time points: immediately prior to the beginning of the production process (fresh buffy coat (FBC); right after the first incubation (incubated buffy coat (IBC)); and at the end of the process thus producing the final product (FP), meaning after hypo-osmotic shock, followed by a second, 90 min incubation with serum at 37° C. At each time point, leukocytes were labeled with specific monoclonal antibodies against corresponding activation markers and then analyzed by flow cytometry.

[0083]For antibody staining, cells from each time point were washed with FACS staining solution (PBS, 2% Normal Mouse Serum; 0.02% Sodium Azide), aliquoted at 0.5×106 / μl and incubated with appropriate monoclonal antibodies for 30 min. at +4° C. in the dark. After incubation, the cells were treated with erythrocyte lysis buffer, washed, re-suspended in PBS and analyzed on FACSCalibur flow cytometer (Becton Dickinson). Anti-CD1...

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Abstract

Disclosed are therapeutic, blood-derived activated leukocyte compositions, methods of making them, and methods of using the compositions to repair or promote the prevention and healing of wounds.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation-in-part of International Application No. PCT / IB2010 / 000882, filed Mar. 5, 2010, in English and designating the United States, which claims benefit of Provisional Application Ser. No. 61 / 209,298, filed Mar. 5, 2009, and Provisional Application Ser. No. 61 / 211,587, filed Apr. 1, 2009, the disclosures of each of which are hereby incorporated herein by reference. This application also claims benefit of Provisional Application Ser. No. 61 / 381,268, filed Sep. 9, 2010, and Provisional Application Ser. No. 61 / 460,024, filed Dec. 23, 2010, the disclosures of each of which are also incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]The wound healing process involves participation of white blood cells, also known as leukocytes. Leukocytes include lymphocytes, granulocytes and monocytes. Three common types of lymphocytes are T-cells, B-cells and natural killer cells. T-cells and B-cells play important...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/14C12N5/07A61K35/12A61K35/15
CPCA61K8/983A61K35/15A61K2035/124A61K35/17A61Q1/145C12N5/0642A61L2300/252A61P17/02A61P31/00A61P43/00C12N5/0645A61L15/44A61L15/40A61L2430/34A61L2300/412
Inventor SHIRVAN, MITCHELLSHINAR, ELLATFRENKEL, ORILZULOFF-SHANI, ADIBUBIS, MARINABAIN, EILATGILLIS, IRENE
Owner MACROCURE